RAD-Induced Nitric Oxide & Cellular Radiosensitivity

描述（由申请人提供）：最近的几项研究表明，电离辐射激活代谢过程，导致活性氧（ROS）的瞬时产生。细胞质电离事件的这种放大和信号传导机制的潜在机制似乎涉及细胞内从一个线粒体传播到另一个线粒体的Ca 2+依赖性信号。时间上一致的是组成性的Ca 2+依赖性一氧化氮合酶（NOS*）的激活。提出了NOS的辐射诱导的活化是一种机制，其（1）限制辐射后形成的细胞毒性、诱变性ROS（02-/H202）的量;和（2）活化参与细胞生长调节的信号转导途径。由于其独特的生理化学性质，辐射后增强的NO生成还提供了由单细胞中发生的电离事件产生的信号在组织内的细胞内和细胞间传播的机制。为了检验这一建议，有两个具体目标： 确定通过分子和药理学手段调节NOS活性是否会增强辐射诱导的细胞杀伤和/或诱变，而这反过来又可以通过超氧化物歧化酶（SOD）的过度表达或用SOD模拟物处理细胞来减少。确定NOS活性的操纵是否调节辐射诱导的ROS产生，测量为02 测试（a）NO-/sGC/PKG信号转导途径的操纵是否改变细胞放射敏感性;（B）辐射刺激的sGC/PKG与下游后果之间的关系，包括c-fos和p21 Waf 1/Cip 1的转录激活和MAPK家族成员的激活，以及（c）辐射刺激的瞬时S-亚硝基化和胞质和核蛋白酪氨酸磷酸酶（PIP）的抑制是否参与细胞对辐射的反应（SHP-2、SHP-1和cdc 25）。

DESCRIPTION (provided by applicant): Several recent studies have demonstrated that ionizing radiation activates a metabolic process resulting in the transient generation of reactive oxygen (ROS). The underlying mechanism for this amplification and signaling mechanism of cytoplasmic ionization events appears to involve a Ca2+ dependent signal propagated from one mitochondria to another within a cell. Temporally coincident is the activation of a constitutive, Ca2+ dependent nitric oxide synthase (NOS*). It is proposed that the radiation-induced activation of NOS is a mechanism that (1) limits the amount of cytotoxic, mutagenic ROS (02-/H202) formed after radiation; and (2) activates signal transduction pathways involved in cell growth regulation. Because of its unique physiochemical properties, the enhanced NO. generation after irradiation also provides a mechanism for the intracellular and intercellular propagation within a tissue of a signal generated by an ionization event that has occurred in a single cell. To test this proposal, there are two specific aims: Determine whether modulation of NOS activity by molecular and pharmacological means enhances radiation-induced cell killing and/or mutagenesis that in turn can be reduced by over expression of superoxide dismutase (SOD) or treating cells with SOD mimetics. Determine whether manipulation of NOS activity modulates radiation-induced ROS generation measured as 02 Test (a) whether manipulation of the NO-/sGC/PKG signal transduction pathway alters cellular radiosensitivity; (b) the relationships between radiation-stimulated sGC/PKG and downstream consequences including transcriptional activation of c-fos and p21 Waf1/Cip1 and activation of members of the MAPK family and (c) whether radiation-stimulated transient S-nitrosylation and inhibition of cytosolic and nuclear protein tyrosine phosphatases (PIP) are involved in cellular responses to radiation (SHP-2, SHP-1 and cdc25).